After trying to clone my first PCR product for almost a year and trying
just about every method I could get my hands on in that time the one that
seems to give me the most consistent and reliable results is quite simple:
I start with a 100 ul Taq polymerase generated PCR product 600 bp - 1.6 kb.
Remove the mineral oil with a chloroform extraction.
Geneclean a portion of the PCR product (~25 ul-50 ul)
Either restriction digest (if you've added sites with your primers) or
blunt it by filling in.
GeneClean a second time.
Then go on to the ligation with this double GeneCleaned product.
Works very well. Just be careful not to carry along any of the glassmilk
or the NewWash as both cause problems with ligation.
You may find that you need to use the "Mermaid kit--Glassfog" for smaller
fragment recovery. Our lab uses both and this seems to be working well
for many of us now.
GeneClean is a product of Bio101--it's just glassmilk.
It's distributed through MidWest Scientific and Research Products International
Hope this helps
Ellen M. Quardokus
ONNO. Myriam (onno.myriam at univ-rennes1.fr) wrote:
: What are the best methods to clone PCR products ?
: What do you think about TA cloning Kit ?
: The size of my insert is 700 bp